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2007; Garc´ıa-S´anchez et al., 2006). Citrus rootstock species control the uptake of chloride (Cl−) and/or sodium (Na+) and the relative tolerance of citrus rootstocks has been based on the accumulation of Cl− in leaves (Levy and Syvertsen, 2004). In Spain, sour orange (Citrus aurantium L.) historically has been an excellent rootstock for citrus trees under saline conditions. However, sour orange is highly susceptible to the tristeza disease (Salibe, 1973) so this rootstock has been replaced by Cleopatra mandarin and Carrizo citrange. Cleopatra mandarin is a good Cl−-excluder whereas ‘Carrizo’ citrange is considered to be a Cl− accumulator but a good Na+ excluder (Ba˜ nuls and Primo-Millo, 1995; Levy et al., 1999). The Cl− restriction mechanism in Cleopatra relative to Carrizo citrange could be linked to either the low absorption of chloride per volume of water, a less efficient root system for water uptake or to a high shoot-to-root ratio (Moya et al., 2003). However, Na+ restriction mechanisms in Carrizo citrange are still unknown. Citrus responses to salinity can depend on growth conditions, amount of irrigation water, climate or soil type (Levy and Syvertsen, 2004; Murkute et al., 2005). For example, under saline conditions, relative yield was increased in Fino 49 lemon on C. macrophylla due to a reduction of the average rootzone salinity by a 25% increase in the amount of water applied (Garc´ıa-S´anchez et al., 2003). Arid or semi-arid climates with large vapor pressure deficits can increase leaf Cl− concentration by increasing foliar transpiration (Moya et al., 1999). In addition, root morphology and ion uptake by roots in solution culture can be different from roots growing in soil (Storey, 1995, Levy and Syvertsen, 2004). There is little information how soil type can influence in the salt tolerance of citrus but soil ion exchange capacity, mechanical impedance to root growth or the effect of soil matric potential could influence the availability of salt ions and thus, change tolerance to salinity (Villagra and Cavagnaro, 2005). In this experiment, we grew the relatively salt-tolerant Cleopatra mandarin and the more salt-sensitive Carrizo citrange citrus rootstock seedlings in four contrasting substrate types: hydroponic solution, river washed sand, perlite, and clay-loam soil. Plant growth responses and leaf and root mineral nutrient status were used as indices of salt tolerance. MATERIALS AND METHODS Plant Material and Growing Conditions Seeds of Carrizo citrange [Citrus sinensis (L.) Osb. × Poncirus trifoliate L., Carr] and Cleopatra mandarin (Citrus reticulata Blanco, Cleo) were germinated in containers containing vermiculite wetted with 0.5 mmol L−1 calcium sulfate (CaSO4 ). When seedlings were two-month-old, they were supported in 1-L containers of continuously aerated Hoagland’s complete nutrient solution, or transplanted into 1-L containers with drainage filled with river

reaching 200◦ C in 20 minutes and holding at this temperature for two hours (Mars Xpress, CEM, Matthews, NC, USA). Electrical Conductivity and Cl− Concentration in the Drainage Solution Electrical conductivity (EC) and Cl− concentration was measured at the end of the experiment in the hydroponic or leached drainage solutions from each pot. The leachate was collected after watering with 50 mL of Hoagland nutrient solution. Electrical conductivity was measured with a Crison EC meter and Cl− concentration was measured with the chloridometer as above.

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Statistical Analysis Data were subjected to analysis of variance using two salinity treatments × four substrates as main effect for each rootstock, and six replicate plants per treatment. When salinity treatment × substrate interactions were significant (P < 0.05), means were separated using Duncan’s multiple range test. RESULTS Growth Overall, Carr seedlings were larger than Cleo seedlings. Plant growth of the non-salinized seedlings was significantly affected by substrate type as total plant dry weight was greatest in seedlings of both rootstocks from solution culture and the lowest in seedlings from clay-loam soil. Salt treatment reduced total plant growth of plants from hydroponic and sand but not of the already smaller plants in perlite and clay-loam soil (Figures 1a, 1c). In Carr seedlings, total plant reductions were about a 29% in both hydroponic and sand, and in Cleo, salinity-induced reductions were about a 24% and 35% for hydroponic and sand, respectively. Carr seedlings in solution culture and sand allocated more growth to shoots than to roots such that their shoot to root dr wt ratios were higher than those from perlite and clay-loam (Figure 1b). Cleo seedlings in sand had the higher shoot to root dw than those from the other substrates (Figure 1d). The salt treatment reduced root and shoot growth similarly as shoot to root dr wt ratio was not affected in Carr or Cleo. Chloride and Sodium Concentration in Leaves and Roots Salinity increased the concentration of Cl− in both leaves and roots of Cleo and Carr seedlings regardless of substrate (Figure 2). In both Carr and Cleo, the highest leaf Cl− concentrations were in seedlings from the clay-loam soil and at least numerically lowest in seedlings from perlite

(Figure 2a). Root Cl− concentration was lowest in seedlings from clay-loam soil in both Carr and Cleo. Concentration of Cl− in Cleo roots was the highest in seedlings from solution culture, whereas perlite-grown seedlings had significantly lower root Cl− than those from sand (Figure 2d). Salinity increased the concentration of Na+ in leaves and roots of both Cleo and Carr seedlings regardless of substrate except in the already low Na+ in Cleo roots (Figure 3). The highest concentration of Na+ in Carr leaves was in seedlings from clay-loam soil and solution culture, and the lowest from those in perlite (Figure 3a). In Cleo, the highest leaf Na+ concentration also was in seedlings from clay-loam soil and the lowest in those from perlite (Figure 3c). Concentration of Na+ in roots was highest in seedlings from solution culture and lowest in roots from clay-loam soil in both Carr and Cleo (Figure 3b, d).

Other Nutrients in Leaves and Roots In the non-salinized control, Carr and Cleo leaves from sand tended to have the highest leaf Ca+2 concentration, and seedlings from perlite and sand tended to have the highest leaf K + concentrations (Table 1). Cleo and Carr seedlings from perlite had the highest leaf Mg2+ concentration while Carr seedlings from solution culture and sand, and Cleo seedlings in solution had the lowest. Carr seedlings from solution culture and sand, and Cleo seedlings from solution culture, had the highest leaf P concentration whereas the lowest concentration was observed in clay-loam soil for Cleo and Carr seedlings. The salt treatment in Carr decreased leaf Ca2+ concentration in seedlings from solution culture and sand. Salinity increased the leaf K + concentration in Carr seedlings from all four substrates, and increased the leaf P concentration in seedlings from perlite and clay-loam soil. In Cleo

seedlings, the salt treatment decreased the leaf Ca2+ concentration in all four substrates, but leaf K +, Mg2+ and P concentration were not affected. Carr roots from non-salinized clay-loam soil and solution culture treatment, and Cleo roots from non-salinized soil treatment had the highest Ca2+ concentration (Table 2). Again, root Ca2+ tended to be lowest in those plants from perlite in both Cleo and Carr. The highest root K + concentration was observed in Carr seedlings from sand and perlite, and in Cleo seedlings from solution culture whereas the lowest K + concentration was observed in clayloam soil for both Cleo and Carr. The highest root Mg2+ concentration was in Carr seedlings from perlite and in seedlings from perlite and clay-loam soil for Cleo. The highest P concentration was in solution culture and the lowest in clay-loam soil for both Cleo and Carr seedlings. In Carrizo, root K + concentration was decreased significantly by salt treatment in seedlings from all four substrates. In Cleo, root Ca2+ concentration was decreased by

Significant differences among substrate × salt treatments are indicated by different lower case letters, whereas within each column, means followed by the same letters are not significant different at P < 0.05. Significant differences between substrate type are indicated by different upper case letters. ns, ∗,∗∗,∗∗∗ indicate non-significant or significant differences at P < 0.05, 0.01, or 0.001 respectively.

salt treatment only in seedlings from sand and clay-loam soil, and root K + concentration was decreased by salt treatment in all four substrates. Electrical Conductivity and Cl− Concentration in Solution The EC and Cl− concentration in the salinized leached solution was highest for clay-loam soil whereas the EC and Cl− in the leachate from perlite and sand was lowest (Figure 4). The EC and Cl− concentration in nutrient solution from solution culture treatment was significantly lower than those of the others substrates. DISCUSSION Effect of Substrate Type without Salinity Both Cleo and Carr seedlings grew the most in solution culture and sand. The fine texture of the compact native clay-loam soil with poor drainage may

Significant differences among substrate × salt treatments are indicated by different lower case letters, whereas within each column, means followed by the same letter are not significant different at P < 0.05. Significant differences between substrate type are indicated by different upper case letters. ns, ∗,∗∗,∗∗∗ indicate non-significant or significant differences at P < 0.05, 0.01, or 0.001 respectively. 16

have negatively affected to the growth in seedlings. The low air-filled porosity could have increased root penetration resistance and reduced oxygen diffusion to the roots (Mcafee et al., 1989). Reduction in soil oxygen (O2 ) concentration from water logging can decrease citrus seedling growth along with stomatal conductance and net assimilation of carbon dioxide (CO2 ) (Garc´ıa-S´anchez et al., 2007), while increasing oxidative damage (Arbona et al., 2008). Drought stress in the perlite substrate also could have limited growth because irrigation every other day may have been inadequate. Perlite has been reported to support excellent growth of tomato seedlings when watered more frequently and maintaining a good water status (Aroiee et al., 2006). These two citrus rootstocks have different abilities to uptake water and nutrients (Castle et al., 1993) as leaves from Carr seedlings had higher Ca2+ and lower K + concentration than leaves from Cleo seedlings. Substrate type also influenced leaf Mg2+ concentration as leaves from perlite, with the highest extractable, had higher leaf Mg+2 concentration than those from sand. However, others factors such as substrate texture which can influence water availability, root production, root to shoot ratio, root morphology and extractable nutrient concentration, could have influenced leaf Ca2+ and K + concentration. For example, clay-loam soil had a greater extractable Ca+ concentration than sand but leaf Ca2+ concentration was similar for Cleo seedlings in both substrates. Leaf Ca2+ concentration in Carr seedlings was higher in sand than in clay-loam soil. We did not evaluate root morphology but second order lateral roots, greater branching and root tip numbers in citrus roots can enhance the capacity to absorb nutrients such as nitrate from nutrient solution (Sorgon`a et al., 2005). Effect of Substrate Type on Salt Tolerance Citrus trees are considered salt sensitive mainly due to the high growth and yield reductions caused by the Cl− and/or Na+ toxicity in the leaves (Garc´ıa-S´anchez et al., 2000). Salinity in the nutrient solution reduced growth in seedlings from solution culture and sand but not in those from perlite or clay-loam soil. However, the highest leaf Cl− concentration was observed in Cleo and Carr seedlings from clay-loam soil. Thus, in this experiment growth reduction by salt treatment could not be linked directly to leaf Cl− and Na+ concentration because physical characteristic of clay-loam soil or drought stress perlite that could have been more limiting to the growth than the effects of salt treatment. Based on absolute values of total plant dry weight from salinized seedlings, the most salt tolerant seedlings were grown in solution culture and the least salt tolerant seedlings were grown in clay-loam soil for both Cleo and Carr. Differences in the leaf mineral nutrient concentrations in the different substrate types could have also influenced salt tolerance. Salinity reduced

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Citrus Seedlings under Saline Conditions

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total plant growth in Carr seedlings from solution culture and sand similarly (38% relative to control treatment) despite the fact that Carr seedlings from sand had higher leaf Cl− concentration (3.13% dw) than those from solution culture (2.20% d.w). Thus, the higher Ca2+ and K + concentration that occurred in leaves from sand could have alleviated the negative effect of the high leaf Cl− concentration. High leaf Ca2+ concentration in citrus ameliorated salt induced leaf abscission so Ca2+ has an intrinsic role in improving growth of salinized plants besides maintaining a balance in Na+/Ca2+ ratio (Romero-Aranda et al., 1998). High leaf Ca2+ concentration in plum increased salt tolerance by maintaining membrane permeability (Bolat et al., 2006). In addition, improving K + nutrition of plants under salt stress can greatly lower ROS production by reducing activity of NAD(P)H oxidases and maintaining photosynthetic electron transport (Cakmak, 2005). In general leaf Cl− concentration was higher in seedlings from Carr and leaf Na+ concentration was higher in seedlings from Cleo supporting the well known idea that Carrizo citrange is considered a Cl− accumulator and Cleopatra mandarin in a Na+ accumulator (Storey and Walker, 1999). In addition, the highest leaf Cl− and Na+ concentration was observed in seedlings from clay-loam soil. In contrast to leaves, the root Cl− and Na+ concentrations tended to be the lowest in seedlings from clay-loam soil suggesting that growing Carr and Cleo seedlings in clay-loam soil decreased the ability of roots to sequester salt ions and thus increased salt accumulation in leaves (Garc´ıa-S´anchez and Syvertsen, 2006). The low leaching fraction of this substrate could have increased the salt ion accumulation as supported by the highest electrical conductivity and chloride concentration in the leachate from salinized clay-loam soil. In addition, the low shoot growth in clay-loam soil could have also increased the leaf Cl− concentration in seedlings by a concentrating effect. It is also possible that some aspect of root growth in the different substrates could have had a direct effect on the Cl− and/or Na+ uptake. Carr and Cleo seedlings grown in sand had higher leaf Cl− concentration than those grown in perlite despite the higher shoot to root ratio in seedlings grown in sand. This too, could have been a function of drought stress in perlite since Cl− accumulation in leaves can be diminished by reducing plant water use (Moya et al., 2003; Garc´ıa-S´anchez et al., 2006). Leaf Ca2+ concentration in Cleo seedlings from all four substrates and in Carr seedlings from solution culture and clay-loam soil was decreased by salinity. Thus, salt treatment also altered mineral nutrient concentration in leaves, although these alterations depended on the seedlings and the type of substrate. Uptake and translocation of Ca2+ from roots to leaves in citrus can be inhibited by salinity (C´amara et al., 2003) and translocation of Na+ to the leaves in C. auratium can lead to a displacement of apoplastic Ca2+ (Zid and Grignon, 1985). Leaf K + concentration was increased by salt treatment in Carrizo citrange seedlings but not in Cleo seedlings. We found similar responses in salinized ‘Sunburst’ mandarin trees grafted on Carrizo citrange

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V. Gimeno et al.

(Garc´ıa-S´anchez et al., 2002). Since Carrizo citrange is a good Na+ excluder from leaves, increasing K + within cells could be a regulatory mechanism to maintain osmotic balance against the high levels of Cl− under saline stress. In seedlings from perlite and clay-loam soil with the lowest leaf P concentration, salinity increased P concentration in leaves of Carr. The increased P accumulation in the shoot is presumably controlled at the root level and salinity can enhance P uptake by roots (Grattan and Grieve, 1992). In conclusion, solution culture and sand media supported more growth in both Cleo and Carr seedlings than the perlite or clay-loam soil substrates. Salt treatment reduced plant growth in solution culture and sand but not in perlite or clay-loam soil, suggesting that plant growth was more limited by the substrate than the salt effect. Seedlings from clay-loam soil had the highest leaf Cl− and Na+ concentration as a consequence of low shoot growth, high Cl−, and Na+ concentration in the growing media and a low ability of the roots to sequester Cl− and Na+. The lowest leaf Cl− and Na+ concentration in seedlings from perlite was perhaps due to decreasing the Cl− uptake by roots. Substrate-mediated growth and nutrient uptake responses can be more important than Cl− uptake in determining salinity tolerance in citrus rootstock seedlings. Comparative studies of relative salt tolerance should consider potential contributions of growth substrate to salinity responses. ACKNOWLEDGMENTS ´ Seneca (Region ´ V. Gimeno is a PhD student supported by the Fundacion de Murcia). Funding for this research came from the Ministerio de Ciencia ´ (Gobierno de Espa˜ e Innovacion na), Project Plan Nacional AGL2007-65437C04-02/AGR. The authors wish to thank to Dr. Walter E. Pereira for the help in the statistical analysis of the data. REFERENCES ´ Arbona, V., Z. Hossain, M. F. Lopez-Climent, R. M. P´erez-Clemente, and A. G´omez-Cadenas. 2008. Antioxidant enzymatic activity is linked to waterlogging stress tolerance in citrus. Physiologia Plantarum 132: 452–466. Aroiee, H., K. Davary, B. Ghahraman, G. A. Peyvast, H. Nematy, and P. Shahinrokhsar. 2006. Effect of different irrigation schedules and substrates on some quantitative and qualitative characteristics of greenhouse tomato (cv. Hamra). Acta Hoticulturae 710: 307–312. Ba˜ nuls, J., and E. Primo-Millo. 1995. Effects of salinity on some citrus scion-rootstock combinations. Annals of Botany 76: 97–102. Bolat, I., C. Kaya, A. Almaca, and S. Timucin. 2006. Calcium sulfate improves salinity tolerance in rootstocks of plum. Journal of Plant Nutrition 29: 553–564. Cakmak, I. 2005. The role of potassium in alleviating detrimental effects of abiotic stresses in plants. Journal and Plant Nutrition and Soil Science 168: 521–530. C´amara, J. M., F. Garc´ıa-S´anchez, M. Nieves, and A. Cerd´a. 2003. Effect of interstock (‘Salustiano’ orange) on growth, leaf mineral composition and water relations of one year old citrus under saline conditions. Journal of Horticultural Science & Biotechnology 78: 161–167. Castle, W. S., D. P. H. Tucker, A. H., Krezdorn, and C. O. Youtsey. 1993. Rootstocks for Florida Citrus. Gainsville, FL: University of Florida, Institute of Food and Agricultural Sciences.